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Disulphide-less crotamine is effective for formation of DNA–peptide complex but is unable to improve bovine embryo transfection

Published online by Cambridge University Press:  30 October 2019

Vicente J.F. Freitas Open the ORCID record for Vicente J.F. Freitas [Opens in a new window] ,
Iana S. Campelo ,
Mirelly M.A.S. Silva ,
Camila M. Cavalcanti ,
Dárcio I.A. Teixeira ,
Luiz S.A. Camargo ,
Luciana M. Melo  and
Gandhi Rádis-Baptista
Vicente J.F. Freitas*
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Iana S. Campelo
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Mirelly M.A.S. Silva
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Camila M. Cavalcanti
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Dárcio I.A. Teixeira
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil
Luiz S.A. Camargo
Affiliation:
Embrapa Dairy Cattle, Juiz de Fora, Brazil
Luciana M. Melo
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza, Brazil Molecular Genetics Research Unit, University Center Fametro (UNIFAMETRO), Fortaleza, Brazil
Gandhi Rádis-Baptista*
Affiliation:
Laboratory of Biochemistry and Biotechnology, Institute of Marine Science, Federal University of Ceará (UFC), Fortaleza, Brazil
*
Authors for correspondence: Vicente J.F. Freitas. State University of Ceará, Av. Dr. Silas Mungusba, 1700 – Fortaleza, CE 60714-903, Brazil. Tel: +55 85 3101 9861. E-mail: [email protected]; Gandhi Rádis-Baptista. Federal University of Ceará, Av. da Abolição, 3207 – Fortaleza – CE 60165-081, Brazil. Tel: +55 85 3366 7000, E-mail: [email protected]
Authors for correspondence: Vicente J.F. Freitas. State University of Ceará, Av. Dr. Silas Mungusba, 1700 – Fortaleza, CE 60714-903, Brazil. Tel: +55 85 3101 9861. E-mail: [email protected]; Gandhi Rádis-Baptista. Federal University of Ceará, Av. da Abolição, 3207 – Fortaleza – CE 60165-081, Brazil. Tel: +55 85 3366 7000, E-mail: [email protected]
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Summary

This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

Keywords

EmbryotoxicityGene deliveryMicroinjectionNucleic acid–peptide complexTransgenesis
Type
Research Article
Information
Zygote , Volume 28 , Issue 1 , February 2020 , pp. 72 - 79
Copyright
© Cambridge University Press 2019 

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